Understanding DNA-Repair Systems That are Fundamental to the Maintenance of Life

The DNA in our body cells is damaged for a
variety of reasons, and thus intercellular DNA-repair systems are
fundamental to the maintenance of life. Now scientists from the UNC
School of Medicine have confirmed and clarified key molecular details of
one of these repair systems, known as nucleotide excision repair.

Using an advanced sequencing technique to map and analyze DNA
damage, the scientists demonstrated the functions in bacterial cells of
two important excision repair proteins: Mfd and UvrD.

"The biochemical mechanisms of these proteins have been known for
years from experiments involving purified protein and DNA, and that's
very important, but in this new work we've clarified these proteins'
roles in living cells," said co-senior author Christopher P. Selby, research assistant professor of biochemistry and biophysics at UNC.

‘Key molecular details of one of the DNA repair systems, known as nucleotide excision repair, has been clarified by researchers.’

"Ultimately, this better understanding of bacterial DNA repair could
be useful toward the development of antibacterial drugs," said
co-senior author Aziz Sancar, the Sarah Graham Kenan Professor
of Biochemistry and Biophysics at UNC.

The research publishes this week in the Proceedings of the National Academy of Sciences.

Sancar was awarded the 2015 Nobel Prize for Chemistry for his
research in the 1980s and early 1990s on excision repair in bacteria and
in human cells. This repair process, which also occurs in animal cells,
fixes one of the most common forms of DNA damage: the bulky adduct, an
unwanted chemical modification of DNA typically caused by a toxin or
ultraviolet (UV) radiation.

To study excision repair in cells, Sancar, Selby and colleagues
recently developed a new technique, XR-seq, which allows investigators
to isolate and sequence the small lengths of adduct-damaged DNA that are
snipped from the genome during the excision repair process. Knowing the
sequences of these DNA snippets allows their locations in the genome to
be mapped precisely. They used this method first in 2015 to generate a
UV repair map of the human genome, and in 2016 they used the XR-seq
method to generate the damage and repair maps of the anticancer
cisplatin drug for the entire human genome. Now they have applied this
method to answer some fundamental questions about damage repair in E. coli with the potential of developing novel antibiotic drugs.

The un-sticker: Mfd

In this study, which was also led by postdoctoral research
associate Ogun Adebali, PhD, the researchers focused largely on Mfd, a
protein known from prior work by Sancar and Selby to have a special -
and mechanistically unusual - role in excision repair in bacteria.

"I think Mfd is the most interesting protein in E. coli,"
Selby said. Here's why: When the DNA of a bacterial gene is being
transcribed into RNA, and the molecular machinery of transcription gets
stuck at a bulky adduct, Mfd appears on the scene, recruits other repair
proteins that snip away the damaged section of DNA, and "un-sticks" the
transcription machinery so that it can resume its work. This Mfd-guided
process is called transcription-coupled repair, and it accounts for a
much higher rate of excision repair on strands of DNA that are being
actively transcribed.

Using XR-seq to map UV-induced damage in E. coli bacteria
cells, the researchers found clear evidence of transcription-coupled
repair in normal cells, but not in cells that lack Mfd, thus confirming
the protein's role in the process.

The unwinder: UvrD

In further experiments, the researchers defined the role of an accessory excision repair protein in E. coli - UvrD, which helps clear away each excised segment of damaged DNA.

In the absence of UvrD, the excised piece of DNA remains bound to
the chromosomal DNA, making it hard for cellular waste-disposal enzymes
to chop it up. At the same time, the repair proteins that excised the
strand tend to remain stuck to it, and are thus kept from moving on to
excise other bits of damaged DNA. UvrD's job is to unwind these damaged
and discarded strands from chromosomal DNA, so that they can be disposed
of quickly and the associated repair proteins can go on to catalyze
additional rounds of repair.

Using XR-seq on UV-damaged E. coli cells, the UNC team
confirmed that without UvrD, excised DNA fragments remain stuck to
chromosomal DNA, survive much longer in cells, and - by holding onto
excision repair proteins - slow down the overall rate of excision repair
in cells.

In addition to clarifying the roles of Mfd and UvrD, the research
generally heralds the use of the new XR-seq technique in mapping and
studying excision repair processes.

"XR-seq provides a new type of sequence data, and in this work we've
provided for the first time a genome-wide map of excision repair in a
bacterium," said Adebali. "We think this map will be broadly useful to
the scientific community."

The researchers now plan further studies using XR-seq in bacterial
cells, as well as in human and other mammalian cells where the process
of excision repair is less understood.

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